Following metabolic stress a variety of gene products are induced in cells in the brain, some of which may protect the tissue from subsequent stresses. The heat shock proteins (hsps), in particular hsp70, have been widely studied in this context, but evidence for the involvement of known hsps in protection of the CNS is inconclusive. We have therefore undertaken the search for other stress-induced proteins which may mitigate ischemic injury. Beginning with degenerate RT-PCR, we have isolated a rat-brain cDNA encoding a protein highly similar to human grp75, a mitochondrial member of the hsp70-family of stress proteins. It is also highly similar to two non-mitochondrial proteins; mortalin, a senescence-related gene product, and pbp74, a protein implicated in B-cell peptide processing. Sequence structure and phylogenetic analyses predict mitochondrial localization and induction by a calcium ionophore and glucose deprivation in PC12 cells support its identification as rat grp75. In situ analysis of normal brain reveals an unusual distribution, with very high expression in neurons of the basal forebrain, reticular and subthalamic nuclei, globus pallidus, amygdala and elsewhere. grp75-mRNA is upregulated following focal brain ischemia in a distinctive fashion. When the degree of injury is small, induction occurs in the area of injury, similar to the pattern observed for hsp70. However, when the injury is extensive, hsr is upregulated in neurons outside the ischemic area. The induction of grp75 may represent a sensitive marker of metabolically compromised tissue.

Fluorosed enamel is more porous and less mineralized, possibly related to altered amelogeninmodulated crystal growth. The purpose of this study was to examine the role of fluoride in interactions between amelogenin and apatite crystals. Recombinant human amelogenin (rh174) was bound to carbonated hydroxyapatite containing various amounts of fluoride, and analyzed by protein assay, SDS PAGE, and AFM. Interactions between rh174 and fluoride were assayed by isothermal titration calorimetry (ITC). The initial binding rate of rh174, as well as total amount of rh174 bound to fluoride-containing carbonated hydroxyapatite, was greater than that in the control carbonated hydroxyapatite. Fluoride in solution at physiologic (5.3 micromolar, or 0.1 ppm) concentrations showed no significant effect on binding, but higher fluoride levels significantly decreased protein binding. ITC showed no interactions between fluoride and rh174. These results suggest that fluoride incorporation into the crystal lattice alters the crystal surface to enhance amelogenin binding, with no direct interactions between fluoride and amelogenin.

A reverse transcriptase-polymerase chain reaction (RT-PCR) product obtained from ischemic rat brain RNA was used to screen a rat ischemic forebrain cDNA library for a cDNA clone containing the entire open reading frame for the inducible hsp70. The coding sequence for the rat hsp70 cDNA demonstrated significant similarities with the human hsp70 of Hunt and Morimoto (Proc Natl Acad Sci 82:6455-6459, 1985) and the mouse hsp70 of Hunt and Calderwood (Gene 87:199-204, 1990). The rat inducible hsp70 and constitutive hsc73 sequences are distinct. There was a low level of hsp70 mRNA expression in normal rat brain as in found in other tissues. hsp70 mRNA was markedly induced in rat brain 8 hours following global ischemia and kainic acid-induced seizures. Northern blots showed a approximately 2.9kb hsp70 mRNA band from control, kainic acid, and ischemic brains. RT-PCR confirmed the presence of hsp70 mRNA in normal rat brain. Since there are at least five human and six mouse inducible hsp70 genes known, many other rat hsp70 genes probably exist that could function in different cells or organelles or be induced under different circumstances.

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